Small animal optical imaging is an increasingly popular technique for studying tumors and other physiological conditions in-vivo in life sciences research and pharmaceutical drug development. Optical imaging techniques can provide low cost, rapid, and quantitative measurements compared to more conventional medical imaging techniques such as MRI, CAT, PET, and SPECT.
Optical imaging techniques typically capture images of a specimen using light in the ultraviolet, visible, and near-infrared (near-IR) regions of the electromagnetic spectrum. It can be difficult, however, to acquire accurate three-dimensional information via optical imaging when tissue, which is a turbid medium, is highly scattering and/or absorbs light at these wavelengths. Some imaging techniques detect spatial distributions of scattered, emitted and transmitted photons (or combinations thereof) emanating from a specimen. Further information about the internal structure of the specimen can be obtained from time-of-flight emission measurements, fluorescence lifetimes, and/or the spectral properties of emitted, scattered, and transmitted photons. In general, many different approaches are known and used for the detection of these photons.
Information about the distribution of light emanating from a specimen can be used as an input to a light diffusion algorithm in order to construct a 3D model of in-vivo entities based on the spatial light distribution, see for example U.S. patent application Ser. No. 10/606,976 entitled “METHOD AND APPARATUS FOR 3-D IMAGING OF INTERNAL LIGHT SOURCES” by Daniel G. Stearns et al., filed on Jun. 25, 2003, the contents of which are incorporated herein by reference. The accuracy of light diffusion algorithms, in general, is enhanced by light distribution information acquired from multiple views of the specimen. In consequence, measurement systems that provide multiple-view capability may be more sensitive and provide higher accuracy than single-view systems.
Systems that capture multiple views of a specimen can do so in various ways. Four techniques for recording multiple views of a specimen are shown in FIGS. 1A-D. FIG. 1A is a schematic diagram of an imaging system where multiple CCD cameras 4 are oriented about a specimen 2 in order to acquire multiple views of the specimen. FIG. 1B is a schematic diagram of a measurement system having a source 6 and a detector 4. Both source 6 and detector 4 are scanned over the surface of specimen 2 in order to capture multiple views of the specimen's surface. The arrows in the figure illustrate the scan directions. FIG. 1C is a schematic diagram of a measurement system that uses multiple mirrors 8 to direct two different views of specimen 2 to two detectors 4. FIG. 1D is a schematic diagram of a measurement system that employs a compound mirror 10 to direct two different side views of specimen 2 to an imaging lens 12, which images these views, along with a front view of specimen 2, to detector array 4.
Three-dimensional information about structures and entities inside living organisms is useful in both research and clinical applications. For example, in pharmaceutical pre-clinical trials, tumors can be grown in immuno-compromised animal models and tracked using imaging techniques such as fluorescence imaging. In some cases, image contrast can be enhanced by labeling entities with molecular fluorophores. The use of labels may also provide information about the internal structure of a specimen, such as the dimensions and/or density of particular internal features that have specific molecular characteristics. Detection of fluorescence emitted by a specimen is a preferred technique because the biochemistry of target-specific fluorescence labeling is well developed.
The advent of genetically engineered cell lines that express fluorescent proteins for in-vivo measurements provides a means to characterize an entity using a unique optical emission signal. These techniques are described, for example, by M. Chalfie et al. in “Green Fluorescent Protein as a Marker for Gene Expression,” Science 263: 802-805 (1994), the contents of which are incorporated herein by reference. Exogenous fluorophores can therefore be introduced into internal structures such as tumors to enable fluorescence imaging by inducing the tumors to express fluorescent proteins, providing for a natural localization of the fluorescence emission within a specimen. In some cases, fluorophores that bind to a tumor can also be injected into a specimen.
The accuracy and resolution of in-vivo imaging of fluorescent entities in living organisms can be limited by scattering and absorption in tissues. These processes attenuate the intensity of light passing through the tissue. The effects of tissue scattering and absorption have been studied extensively, see for example T. L. Troy and S. N. Thennadil, “Optical properties of human skin in the near infrared wavelength range of 1000 to 2200 nm,” Journal of Biomedical Optics 6: 167-176 (2004), the contents of which are incorporated herein by reference. At wavelengths from the ultraviolet to the near-infrared, it has generally been found that as the wavelength of incident light increases, both scattering and absorption of the incident light by tissue decrease. As a result, the effective “penetration depth” of incident light in tissue varies with wavelength. In the range of wavelengths from about 400 nm to about 1100 nm, the greatest penetration depth occurs at about 800 nm.
Absorption and scattering properties of biological tissues have been found to be substantially similar among animals, and this finding has been used as a basis for numerous optical techniques for probing turbid media with light. Computational techniques for reconstructing 3D tissue models that take into account measured spatial, temporal, and/or spectral information from light emitted or scattered by specimen tissues are used to visualize the specimen's internal structure. Monte Carlo methods may be used to validate these structural models, see for example Q. Liu, C. Zhu and N. Ramanujam, “Experimenal validation of Monte Carlo modeling of fluorescence in tissues in the UV-visible spectrum,” Journal of Biomedical Optics 8: 223-236 (2003).
Depth or position information regarding entities within a specimen can be provided by measuring wavelength shifts of light emitted from the specimen, because the magnitude of the wavelength shift varies as a function of the thickness of tissue between the emitting entity and a detector. In particular, if a specimen emits light in a portion of the spectrum where the scattering and/or absorption properties have a substantially monotonic increase or decrease as a function of tissue thickness, then the emission spectrum of a labeled entity will shift as a function of the entity's position within the specimen. The shift of the emission spectrum may be small, i.e., a few nanometers, for significant changes in tissue thickness, so measurement equipment used to detect spectral shifts should have sensitivity that is sufficient to detect small spectral changes in order to make precise thickness estimates. Further, shifts of the emission spectrum can be produced via other mechanisms such as biochemical processes.